The present invention refers to a microfluid device and a method of producing diffusively built gradients, particularly in the field of microfluidics.
Concentration gradients of certain substances in liquid media are of decisive meaning in many applications. It is for instance known that a defined pH gradient is required for the isoelectric focusing of proteins.
In the field of cytology, defined concentration courses are of decisive meaning for research. The precise concentration for instance, at which a substance acts toxically on a cell culture, can be determined in that a continuous concentration increase of the substance to be examined is built up in the culture medium over a homogeneous cell layer.
A defined and long-term stable (over several hours to days) gradient is also required to examine the chemotactic behavior of slow cells, e.g. in that the migration of cells is observed at a mean velocity of e.g. 20 μm/h in or against the direction of a concentration gradient. Some differentiations of cells in living organisms have for instance the capability of moving in the direction of the source (point or space of high concentration compared to the depression, a point or space of low concentration) of determined substances, which is generally termed as “chemotaxis”. In this manner, leucocytes may for instance accumulate at foci of inflammation and vascular precursor cells may form new vessels in the regions which are undersupplied for instance with oxygen or nutriments. The prevention of this mechanism is a promising approach in the control of rapidly growing tumors. It could be attempted to prevent the supply with nutriments and oxygen in excrescent tissue. Furthermore, chemotaxis of tumor cells plays an important role in metastasis. Thus, particularly quantitative measurements of the chemotactic behavior of cells of the human body are of interest, which move at a speed of approx 20 μm per hour towards the source of the messenger.
In order to obtain significant data it is assumed that preferably paths of cells of approximately 20 times the cell diameter are to be observed. Typical cell diameters are between 5 μm and 30 μm. To cover for instance 600 μm, a cell with a mean velocity of 20 μm/h needs 30 hours, which corresponds to a typical observation period.
The prior art concerned with the structure of a concentration gradient will now be explained by means of the structure in experiments for chemotactic examinations.
The chamber developed by Boyden (Boyden, S., 1962: “The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes”, J Exp Med 115: 453-466) has been in use in various forms up to the present day. In this system a porous membrane separates two chambers in which different concentrations of chemokines are located. A step-like to sigmoid concentration gradient forms in the membrane area. The cells are applied onto one side of the membrane and actively move to the other side in that they migrate through the pores. After a determined period of time, the chamber is removed, the filter is removed and the cells are counted after a dyeing step on the upper and lower side of the membrane.
The difficulty of the evaluation is the distinction between chemotaxis and increased random movement (chemokinese). It may also happen that migrated cells detach from the membrane surface, thus falsifying the result. Only by means of parallel supervision can it be verified whether the cells have actually reacted on stimulus and have therefore migrated or whether they merely randomly moved to the other side of the membrane. Microscopic observations of the cells during the experiment are not possible.
A further method is the use of microcapillaries. In this system a microcapillary with a microscopically small opening is moved in the proximity of the cells that are located in a cell vessel. The opening of the capillaries and the medium surrounding the cells are fluidically connected to one another (Gerisch and Keller, 1981). The chemokine diffuses or flows out of the opening of the capillaries that is located in the proximity of the cell or a cell compound.
The capillaries are of glass and must therefore be handled with care. Furthermore, a micro-manipulator is required for handling the capillaries in the proximity of the cells. A further disadvantage of this system is the high costs and the inconvenience in application. Moreover, the radial form of the gradient around the capillary opening is only suitable for the simultaneous observation of single or few cells. The gradient may be adjusted in a locally very steep manner. The form and time response of the gradient are complex and cannot be quantified. Smaller flows within the cell culture vessel also lead to massive concentration changes that cannot be quantified. Such flows may for instance be caused by convection. Quantitative statements about the migration behavior of many cells are generally possible by several parallel experiments only, which, however cannot be carried out identically due to the complex handling and which can therefore not be compared to one another. Furthermore, image processing is required for the evaluation of the data.
The Zigmond chamber (Zigmond, S. H., 1977: “Ability of polymorphonuclear leukocytes to orient in gradients of chemotactic factors”, J Cell Biol 75: 606-16; Zigmond, D. H., 1988: “Orientation chamber in chemotaxis”, Methods in enzymology 162: 65-72) is composed of two chambers that are separated between the chambers by a thin observation volume. The observation volume is restricted from the top by a cover glass, which is mounted by suitable mechanical attachment at a defined distance from the surface. The water level in the two chambers must possibly precisely correspond to the height of the cover glass. Identical cell culture medium is located in both chambers, wherein a defined amount of the chemokine is added to one of the chambers. A linear chemical gradient is formed by diffusion in the ideal case. The use of the cover glass enables to microscopically follow the chemotactic movement of the cells.
A restriction for such examinations is that cells can only react to gradients if the concentration drop over a cell length is approximately 1% of the mean concentration at the location of the cell. That means that the gradient must be sufficiently steep. By diffusion and undesired flow the gradient in real Zigmond chambers is for approximately 30 to 60 minutes sufficiently steep for chemotaxis studies. After that it is too flattened or interfered by flows. Moreover, the application of the Zigmond chambers demands great skill in the performance of the respective tests, since liquid flows can quickly destroy the gradient due to the multipiece structure and the open chambers.
The Dunn chamber (Zicha, D., G. A. Dunn and A. F. Brown, 1991: “A new direct-viewing chemotaxis chamber”, J Cell Sci 99 (Pt 4): 769-75) is a further development of the Zigmond chamber. The two portions that contain or not contain the chemokine are arranged radially with respect to one another and are separated from one another by a closed, annular barrier. After putting on the cover glass, the gap serving for examination is located between the annular barrier and the cover glass so that it can be examined whether the cells extend in an aimed manner e.g. towards the center of the arrangement if the chemokine is added in the central portion.
The Dunn chamber allows the chemotactic examination over longer periods of time than the Zigmond chamber. The geometry in this case is disadvantageous, since the direction of the chemotactic movement to be expected depends on the location within the gap. This makes the valuation of the data more complex. Moreover, the chemical gradient is very instable compared to mechanical influences, such as holding the chamber at an oblique angle, which amongst others leads to an interference of the diffusive gradient caused by the additionally occurring flow.
The chemotactic behavior of the respective cell type does generally not only depend on the substance but also on its concentration and steepness of the concentration decline. The standard value for the concentration decline that can just about be detected by the cell may be—as already mentioned above—about 1% decline per cell length at the location of the cell.
Chemotactically active cells, such as human umbilical vein endothelial cells (HUVEC), which amongst others react to the substance VEGF (vascular endothelial growth factor) and the tumor cell line HT1080, which chemotactically reacts to FCS (fetal calf serum) serve as an example for cells.